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- EMDB-2834: Cryo-EM structure of TMV reconstructed from micrographs recorded ... -

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Basic information

Entry
Database: EMDB / ID: EMD-2834
TitleCryo-EM structure of TMV reconstructed from micrographs recorded with the Falcon II DED
Map dataCryo-EM structure of TMV reconstructed from micrographs recorded with the Falcon II DED
Sample
  • Sample: Tobacco Mosaic Virus
  • Virus: Tobacco mosaic virus
Keywordstobacco mosaic virus / direct electron detectors / single particle helical reconstruction
Biological speciesTobacco mosaic virus
Methodhelical reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsFromm SA / Bharat TAM / Jakobi AJ / Hagen WJH / Sachse C
CitationJournal: J Struct Biol / Year: 2015
Title: Seeing tobacco mosaic virus through direct electron detectors.
Authors: Simon A Fromm / Tanmay A M Bharat / Arjen J Jakobi / Wim J H Hagen / Carsten Sachse /
Abstract: With the introduction of direct electron detectors (DED) to the field of electron cryo-microscopy, a wave of atomic-resolution structures has become available. As the new detectors still require ...With the introduction of direct electron detectors (DED) to the field of electron cryo-microscopy, a wave of atomic-resolution structures has become available. As the new detectors still require comparative characterization, we have used tobacco mosaic virus (TMV) as a test specimen to study the quality of 3D image reconstructions from data recorded on the two direct electron detector cameras, K2 Summit and Falcon II. Using DED movie frames, we explored related image-processing aspects and compared the performance of micrograph-based and segment-based motion correction approaches. In addition, we investigated the effect of dose deposition on the atomic-resolution structure of TMV and show that radiation damage affects negative carboxyl chains first in a side-chain specific manner. Finally, using 450,000 asymmetric units and limiting the effects of radiation damage, we determined a high-resolution cryo-EM map at 3.35Å resolution. Here, we provide a comparative case study of highly ordered TMV recorded on different direct electron detectors to establish recording and processing conditions that enable structure determination up to 3.2Å in resolution using cryo-EM.
History
DepositionDec 9, 2014-
Header (metadata) releaseDec 24, 2014-
Map releaseDec 24, 2014-
UpdateMar 11, 2015-
Current statusMar 11, 2015Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.2
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 1.2
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 1.2
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_2834.png

Downloads & links

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Map

FileDownload / File: emd_2834.map.gz / Format: CCP4 / Size: 31.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM structure of TMV reconstructed from micrographs recorded with the Falcon II DED
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.06 Å/pix.
x 190 pix.
= 201.59 Å		1.06 Å/pix.
x 210 pix.
= 222.81 Å		1.06 Å/pix.
x 210 pix.
= 222.81 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 1.061 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 1.2 / Movie #1: 1.2
Minimum - Maximum-4.22035074 - 5.3352294
Average (Standard dev.)0.01439038 (±0.75744009)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-105-105-95
Dimensions210210190
Spacing210210190
CellA: 222.81 Å / B: 222.81 Å / C: 201.59 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0611.0611.061
M x/y/z210210190
origin x/y/z0.0000.0000.000
length x/y/z222.810222.810201.590
α/β/γ90.00090.00090.000
start NX/NY/NZ-40-32-96
NX/NY/NZ8165193
MAP C/R/S123
start NC/NR/NS-105-105-95
NC/NR/NS210210190
D min/max/mean-4.2205.3350.014

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Supplemental data

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Sample components

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Entire : Tobacco Mosaic Virus

EntireName: Tobacco Mosaic Virus
Components
  • Sample: Tobacco Mosaic Virus
  • Virus: Tobacco mosaic virus

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Supramolecule #1000: Tobacco Mosaic Virus

SupramoleculeName: Tobacco Mosaic Virus / type: sample / ID: 1000 / Details: The sample was purified from infected leaves. / Oligomeric state: helical / Number unique components: 1
Molecular weightTheoretical: 39.5 MDa

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Supramolecule #1: Tobacco mosaic virus

SupramoleculeName: Tobacco mosaic virus / type: virus / ID: 1 / Name.synonym: TMV / NCBI-ID: 12242 / Sci species name: Tobacco mosaic virus / Sci species strain: vulgare / Database: NCBI / Virus type: VIRION / Virus isolate: STRAIN / Virus enveloped: No / Virus empty: No / Syn species name: TMV
Host (natural)Organism: Nicotiana tabacum (common tobacco) / synonym: PLANTAE(HIGHER PLANTS)
Molecular weightTheoretical: 39.5 MDa
Virus shellShell ID: 1 / Name: CP / Diameter: 180 Å

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

Concentration5.5 mg/mL
BufferpH: 7 / Details: 50 mM NaPO4
GridDetails: Quantifoil 2/2 Cu 300 mesh grids
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 95 K / Instrument: FEI VITROBOT MARK IV / Details: Blotting at 283 kelvin
Method: Blot for 2.5 seconds with force -15 ~10 seconds after sample application and with a drain time of 0.5 seconds.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Alignment procedureLegacy - Astigmatism: CTFTILT
DetailsNanoprobe mode, dose rate ~50 e-/px/s on the camera level
DateMar 14, 2014
Image recordingCategory: CCD / Film or detector model: FEI FALCON II (4k x 4k) / Number real images: 28 / Average electron dose: 43 e/Å2
Details: each micrograph was recorded as a movie of 18 frames including the roll-in and roll-out frame which were discarded for image processing
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 131951 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 75000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

DetailsImage processing was done using SPRING.
Final reconstructionApplied symmetry - Helical parameters - Δz: 1.408 Å
Applied symmetry - Helical parameters - Δ&Phi: 22.03 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 3.7 Å / Resolution method: OTHER / Software - Name: SPRING
Details: final map was calculated with the dose limited to 21.5 e-/A^2 and with segment-based motion correction
CTF correctionDetails: CTFTILT (specific for each segment)
Final angle assignmentDetails: SPIDER
FSC plot (resolution estimation)

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